**4. Factors affecting the generation of methane**

Anaerobic microorganisms, especially methanogens are highly susceptible to changes in environmental conditions. Many researchers evaluate the performance of an anaerobic system based on its methane production rate because methanogenesis is regarded as a ratelimiting step in anaerobic treatment of wastewater. Methanogens are highly vulnerable and extremely low growth rate in an anaerobic treatment system require careful maintenance and monitoring of the environmental conditions. A temperature change in the substrates or substrates concentration can lead to shutdown of gas production (Novaes, 1986).

The microbial metabolism processes are dependent on many parameters, so that for an optimum fermenting process, numerous parameters must be taken into consideration and be controlled. Some of these environmental conditions are shown in the Table 1 (Deublein and Steinhauser, 2008). A brief discussion of the factors more reported in literature is shown follows.


Table 1. Environmental conditions and inhibitors in the degradation methanogenic (Deublein and Steinhauser, 2008).

#### **4.1 Temperature**

It is interesting to note that anaerobic digestion in the natural environments occurred in a wide range of temperatures between 4 ºC (lake sediment) to 60 ºC (thermophilic digestion process); however, for the industrial practices, the temperature range is limited to 20-55 ºC

methane generation in an anaerobic treatment system. It bears mentioning that there are many H2-using methanogens that can use formate as a source of electrons for the reduction

4HCOO- + 2H+ CH4 + CO2 + 2HCO3- (6)

Anaerobic microorganisms, especially methanogens are highly susceptible to changes in environmental conditions. Many researchers evaluate the performance of an anaerobic system based on its methane production rate because methanogenesis is regarded as a ratelimiting step in anaerobic treatment of wastewater. Methanogens are highly vulnerable and extremely low growth rate in an anaerobic treatment system require careful maintenance and monitoring of the environmental conditions. A temperature change in the substrates or

The microbial metabolism processes are dependent on many parameters, so that for an optimum fermenting process, numerous parameters must be taken into consideration and be controlled. Some of these environmental conditions are shown in the Table 1 (Deublein and Steinhauser, 2008). A brief discussion of the factors more reported in literature is shown

**Operation Parameters Inhibitors** 

Disintegration Ammonium (NH4+) and ammonia (NH3)

Temperature Disinfectants, herbicides and insecticides Alkalinity and pH Degree of decomposition of organic matter

Table 1. Environmental conditions and inhibitors in the degradation methanogenic

It is interesting to note that anaerobic digestion in the natural environments occurred in a wide range of temperatures between 4 ºC (lake sediment) to 60 ºC (thermophilic digestion process); however, for the industrial practices, the temperature range is limited to 20-55 ºC

(fatty acids and amino acids)

substrates concentration can lead to shutdown of gas production (Novaes, 1986).

de CO2 to methane, as show in reaction (Eq. 6):

follows.

Trace elements Precipitants

Biogas removal

**4.1 Temperature** 

(calcium carbonate, MAP, apatite)

(Deublein and Steinhauser, 2008).

**4. Factors affecting the generation of methane** 

Hydrogen partial pressure Oxygen (O2) Concentration of the microorganisms Sulfur compounds Type of substrate Organic acids

Specific surface of material Nitrate (NO3-)

Cultivation, mixing and volume load Heavy Metals Light and Mixing Tannins

Organic Loading Rate (OLR) Foaming Nutrients (C/N/P-ratio) Scum

(Fannin, 1987). In the natural environments, the optimum temperature for the growth of methane forming *archaea* is 5-25 ºC for psychrophilic, 30-35 ºC, for mesophilic, 50-60 ºC, for thermophilic and >65 ºC for hyprethermophilic (Tchobanoglous and Burton, 1996).

It is generally understood that higher temperature could produce higher rate of reaction and thus promoting higher application of organic loading rate (OLR) without affecting the organic removal efficiency (Chae et al., 2007; Choorit and Wisarnwan, 2007; Poh and Chong, 2009). Using palm oil mill effluent as the substrate, Choorit and Wisarnwan (2007) demonstrated that when the digester was operated at thermophilic temperature (55 ºC), showed higher OLR application than the that of mesophilic (17.01 against 12.25 g COD/ m3-d) and the methane productivity was also higher (4.66 against 3.73 L/L/d) (Choorit and Wisarnwan, 2007). A similarly study by Chae et al (2007), indicated that the higher temperature of 35 ºC led to the highest methane yield as compared to 30 ºC and 25 ºC although the methane contents only changed slightly.

Using cheese whey, poultry waste and cattle dung as substrates, Desai et al. (1994) showed that when the temperature was increased from 20, 40 and 60 ºC, the biogas production and methane percentage increased as well. The digestion rate temperature dependence can be expressed using Arrhenius expression:

$$r\_t = r\_{30} \text{(1.11)}^{(t-30)} \tag{7}$$

where *t* is temperature in ºC, and *rt*, *r30* are digestion rates at temperature *t* and 30ºC, respectively. Based in Eq. 7, the decrease in digestion rate for each 1 ºC decreased in temperature below the optimum range is 11%. Similarly, the calculated rate at 25 ºC y 5 ºC are 59 and 7% respectively, relative to the rate at 30 ºC (Dasai et al., 1994).

Although the thermophilic anaerobic process could increase the rate of reaction, the yield of methane that could be achieved over the specified organic amount is the same regardless of the mesophilic or thermophilic conditions. That value is 0.25 kg CH4/kg COD removed or 0.35 m3 CH4/kg COD removed (0 ºC, 1 atm) which is derived by balancing the following equation (Eq. 8), taking into account the different operating conditions worked, can be explained that the values obtained for methane production is different in many scientific reports:

$$\text{CH} + 2\text{O}\_2 \rightarrow \text{CO}\_2 + 2\text{H}\_2\text{O} \tag{8}$$

Although thermophilic condition could result in higher application of organic loading rates and better destruction of pathogens, at the same time it is more sensitive to toxicants and temperature control is more difficult (Gerardi, 2003; Choorit and Wisarnwan, 2007). Furthermore, biomass washout that could lead to volatile fatty acids accumulation and methanogenesis inhibition could also occur if the thermophilic temperature could not be controlled (Poh and Chong, 2009). As a result, in tropical regions mesophilic temperatures are the preferred choice for anaerobic treatment (Yacob et al., 2005, Sulaiman et al., 2009).

#### **4.2 Alkalinity and pH**

As far as the anaerobic digestion process is concerned, it is more appropriate to discuss alkalinity and pH together because these parameters are related to each other and very

Biogas Production from Anaerobic Treatment of Agro-Industrial Wastewater 99

"known as biomass wash-out" are observed in the effluent, indicating that the reactor suffered a process imbalance and that biomass accumulated in the reactor (Converti et al., 1993; Fezzani and BenCheikh, 2007; Rincón et al., 2008). This could be ascribed to an increase in the concentrations of the VFA with a consequent decrease in pH (Tiwari et al., 2006) or to

Therefore, there is a maximal operational value for this parameter. For instance, Rizzi and coworkers in the year of 2006 reported a decrease in COD removal and specific methane production when OLR was increased from 10 to 15 kg COD/m3-d. With the OLR increase to 20 kg COD/m3-d the biomass excess started to wash out, followed by deterioration of the reactor performance. In a different study, stable reactor performance was observed when the OLR increased from 1.5 to 9.2 kg COD/m3-d with the maximum methane production rate achieved for an OLR of 9.2 kg COD/m3-d. However, a significant decrease in the pH value (from 7.5 to 5.3) was observed when OLR was further raised to 11.0 kg COD/m3-d. In addition, the increase in the effluent COD with increased OLR was paralleled to a sharp increase in the effluent total volatile fatty acids (TVFA, g acetic acid/L) by about 400% (Rincón et al., 2008). This indicates that, at higher OLR the effluent total COD and mainly soluble COD is largely composed of the unused volatile acids produced in the reactor due to

*Methanobacteriaceae* and *Methanosaeta* were found the main methanogens in a laboratory scale up-flow anaerobic digester treating olive mill wastewater (Rizzi et al., 2006). However, the authors also reported an interesting population shift by OLR variation. At lower OLR i.e. 6 kg COD/m3-d, hydrogenotrophic Methanobacterium predominated in the reactor but the number of cells/g sludge showed a 1000 fold decrease from 1011 to 108 when the OLR was increased to 10 kg COD/m3-d. In contrast, phylotypes belonging to the acetoclastic Methanosaeta were not affected by OLR variation and at 10 kg COD/m3-d, dominated in

Olive oil wastewater is characterized by high levels of inhibitory compounds such as tannins, and lipids. As a result, increased OLR leads to higher concentration of these substances and a consequent inhibition of methanogenic cells. However, acetoclastic *Methanosaeta* due to its high affinity for acetate is capable of occupying the deepest and thus more protected niches in the granule or biofilm with low concentrations of substrate (acetate) (Gonzales-Gil et al., 2001). Phylotypes belonging to the genus *Methanosaeta* were also dominant independent of different

In a different study was investigated the microbial ecology of granules in UASB reactor fed by synthetic wastewater under various OLR. The authors showed that the predominant microbial biomass was *Methanosaeta*. However, increasing the OLR led to a substantial increase of *Methanosarcina* in the granules (Kalyuzhnyi et al., 1996). The increase of *Methanosarcina* in the studied synthetic wastewater (toxin-free) due to increasing OLR is explained by the low affinity of these methanogens for acetate in comparison with *Methanosaeta*. Hence, by

As reviewed earlier, under mesophlic conditions *Methanosaeta* plays a significant role in making cores of sludge granules (Sekiguchi et al., 2001) and thus their ratio seems to control the speed of granulation (Rincón et al., 2008). Higher OLR, result in consequent higher concentration of substrates (i.e. acetate) in the reactor. Morvai and coworkers in 1990

increasing OLR and consequent VFA concentration, *Methanosarcina* is favored.

escalated levels of inhibitory or toxic compounds such as phenols, lignin and others.

the inhibition of methanogenesis.

the biofilm (109 cells/g sludge) (Rizzi et al., 2006).

OLR in other anaerobic digesters (Rincón et al., 2008).

promising to ensure a suitable environment for successful methanogenesis process. Alkalinity is produced in the wastewaters as results of the hydroxides and carbonates of calcium, magnesium, sodium, potassium or ammonia and may also include borates, silicates and phosphates (Tchobanoglous and Burton, 1991). The alkalinity plays an important pH controlling role in the anaerobic treatment process by buffering the acidity derived from the acidogenesis process (Gerardi, 2003; Fannin, 1987).

2009) and could only survive on a very narrow range of pH (Table 2) (Gerardi, 2003). Genus pH Range

Methane producing methanogens are known to be strongly affected by pH (Poh and Chong,


Table 2. The optimum pH range for selected methanogens (Gerardi, 2003; Steinhaus et al.2007, Tabatabaei et al., 2011)

As such, the methanogenic activity will be severely affected once the optimum pH range is not met. Steinhaus and coworker studied the optimum growth conditions of *Methanosaeta concilii* using a portable anaerobic microtank (Steinhaus et al., 2007). They reported an optimum pH level of 7.6 revealing that even little variations on both sides of the optimum pH suppressed the growth of the methanogens. Several studies have also reported reactor failure or underperformance simply due to pH reduction caused by accumulation of high volatile fatty acids in the anaerobic treatment system (Fabián and Gordon, 1999; Poh and Chong, 2009; Tabatabaei et al., 2011).

In a study using synthetic wastewater in the thermophilic temperature, was found that at the pH of above 8.0, the methanogenesis was strongly inhibited and the value recorded for acetotrophic methanogenic test was zero (Visser et al., 1993). When investigating the role of pH in anaerobic degradation test; Fabián and Gordon (1999), found out that the acidification led to the low performance of the anaerobic degradation, however the biodegradation was significantly increased once the wastewater when the pH was adjusted to above 6.5.
